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The Osteogenic Effect of Erythropoietin on Human Mesenchymal Stromal Cells is Dose-Dependent and Involves Non-Hematopoietic Receptors and Multiple Intracellular Signaling Pathways

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Abstract

Erythropoietin (EPO) is a pleiotropic growth factor. Of interest for skeletal tissue engineering, the non-hematopoietic capabilities of EPO include its osteogenic and angiogenic potencies. The main aim of this study was to investigate the dose–response relationship and determine the lowest effective dose of EPO that reliably increases the osteogenic differentiation of human mesenchymal stromal cells (hMSCs). Additional aims were to elucidate the surface receptors and to investigate the role of the intracellular signaling pathways by blocking the mammalian target of rapamycin (mTOR), Jak-2 protein tyrosine kinase (JAK2), and phosphoinositide 3-kinases (PI3K). The primary outcome measures were two mineralization assays, Arsenazo III and alizarin red, applied after 10, 14, and 21 days. Moreover, alkaline phosphatase activity, cell number, and cell viability were determined after 2 and 7 days. A proportional dose–response relationship was observed. In vivo, the lowest effective dose of 20 IU/ml should be used for further research to accommodate safety concerns about adverse effects. Ex vivo, the most effective dose of 100 IU/ml could facilitate vascularization and bone ingrowth in cell-based scaffolds. The expression of non-hematopoietic receptors EPOR and CD131 was documented, and EPO triggered all three examined intracellular pathways. Future studies of the efficacy of EPO in cell-based tissue engineering can benefit from our findings.

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Acknowledgments

We express our sincere gratitude to FACS Core Facility at Aarhus University, Denmark, for performing flow cytometry.

Conflicts of interest

The authors declare no potential conflicts of interest.

Financial support

We would like to extend our appreciation to the VELUX foundation for being the main financial contributor of this study. The Helga and Peter Kornings Foundation and Rygforskningsfonden i Aarhus granted additional financial support.

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Authors and Affiliations

  1. Orthopaedic Research Laboratory, Aarhus University Hospital, Aarhus, Denmark, Noerrebrogade 44, Building 1A, 1.tv, 8000, Aarhus, Denmark

    Jan Hendrik Duedal Rölfing, Anette Baatrup, Jonas Jensen, Helle Lysdahl & Cody Bünger

  2. University Centre for Orthopaedics and Traumatology and Centre for Translational Bone, Joint and Soft Tissue Research, University Hospital Carl Gustav Carus at Technische Universität Dresden, Dresden, Germany

    Maik Stiehler

  3. Department of Orthopaedics, Aarhus University Hospital, Aarhus, Denmark

    Jan Hendrik Duedal Rölfing & Cody Bünger

Authors
  1. Jan Hendrik Duedal Rölfing
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  2. Anette Baatrup
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  3. Maik Stiehler
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  4. Jonas Jensen
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  5. Helle Lysdahl
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Correspondence to Jan Hendrik Duedal Rölfing.

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Figure S1

Dose-response relationship and lowest effective dose of EPO, related to Fig. 1. Absolute values comparing EPO [IU/ml] groups with osteogenic medium (Pos C) are depicted for the following outcome measures: cell viability assay (XTT), alkaline phosphatase activity (ALP), and mineralization assay Arsenazo III (Arsenazo). Cells were continuously stimulated with EPO. Proliferation medium was employed as negative control (Neg C). Data are presented as mean ± SD and post-hoc p-values against Pos C are given. (PDF 95 kb)

Figure S2

Intracellular pathway analyses, related to Fig. 5. Outcome measurements: cell viability (XTT), alkaline phosphatase activity (ALP), and primary outcome mineralization assays alizarin red (AZR) and Arsenazo III (Arsenazo) presented as absolute values. In these experiments, all hMSCs were continuously stimulated with 20 IU/ml EPO. Potential intracellular pathways were screened by constant blocking of mTOR with 10 nM rapamycin (Rapa), JAK2 with 10 μM AG490, and PI3K with 50 μM LY294002 (LY) or 50 nM wortmannin (Wort). Data are presented as mean ± SD. * p < 0.0001, ** p < 0.001. (PDF 108 kb)

Figure S3

Intracellular pathway analysis alizarin red staining after 14 days, related to Fig. 5. Representative alizarin red staining visualizing mineralization after 14 days continuous stimulation with 20 IU/ml EPO with and without inhibition with intracellular pathway inhibitors rapamycin, AG490, LY4229002 or Wortmannin. Proliferation medium was employed as negative control (Neg C). (PDF 1034 kb)

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Rölfing, J.H.D., Baatrup, A., Stiehler, M. et al. The Osteogenic Effect of Erythropoietin on Human Mesenchymal Stromal Cells is Dose-Dependent and Involves Non-Hematopoietic Receptors and Multiple Intracellular Signaling Pathways. Stem Cell Rev and Rep 10, 69–78 (2014). https://doi.org/10.1007/s12015-013-9476-x

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